We devised a versatile intrathecal injection method enabling evaluation of central nervous system (CNS) toxicities in rats, and then explored appropriate physico-chemical properties for injectable test solutions at relatively large dosage volumes. In a preliminary bolus injection study, a 25-gauge needle was inserted into the subarachnoid space through the lateral aspect of the intervertebral portion between the 2nd and 3rd lumbar vertebra under light ether anesthesia. When 3% phthalocyanine blue solution, a vital dye, was injected in order to confirm its transfer to the brain tissue, the dye reached the cerebral ventricle, perivascular spaces and veins of the cerebral cortex. On the basis of these information, a fine polyethylene tube was introduced up to a level around the axis via a 20-gauge needle inserted in advance into the vertebral space by the same way. The needle was then withdrawn from the space leaving the tube filled with a test solution. After a full recovery from anesthesia, the infusion was commenced. Injection volume-versus-speed or osmolality-versus-pH relationship was assessed using solutions with various compositions, followed by monitoring mortality coupled with clinical signs. The tolerable combination of factors for the intrathecal solution without causing death was thought to be an injection volume up to 2 ml/rat with 300 mOsm/kg H2O, pH 3 to 7, at a injection speed of less than 0.5 ml/min, although minor clinical signs were observed. Pathological examination revealed pulmonary edema in dead animals, but no changes in surviving animals. This method applicable to conscious rats is considered to be simple and reliable, and does not require surgical operation and special equipment. The toxicological event in the intrathecal route seems to depend largely on the physico-chemical characteristics of the injectable solution, under these experimental conditions.